Excretory-Secretory Antigens of Trypanosoma cruzi Are Potentially Useful for Serodiagnosis of Chronic Chagas' Disease

The reactivities of sera from chronic chagasic patients against the trypomastigote excreted-secreted antigens (TESA) of Trypanosoma cruzi strains with different biodemes were analyzed by TESA-blot and TESA–enzyme-linked immunosorbent assay (ELISA). Although both tests presented high sensitivity and specificity, TESA-ELISA is more appropriate for screening a larger number of samples.     

Chagas' heart disease in the United States

BACKGROUND AND METHODS. Chagas' heart disease is believed to be rare in the United States, although many persons from countries where the disease is endemic reside here. We performed a retrospective case review and prospective follow-up of 25 patients with Chagas' heart disease and no obstructive coronary artery disease on angiography. RESULTS. The patients mainly presented with symptomatic atrioventricular block, congestive heart failure, anginal chest pain, sudden death averted by resuscitation, or sustained ventricular tachycardia. Of the 25 patients, 18 had been treated for coronary artery disease or idiopathic dilated cardiomyopathy for up to 108 months before the diagnosis of Chagas' disease was considered. The electrocardiograms frequently suggested coronary artery disease. Six of the seven patients who had exercise thallium-perfusion scans had abnormalities suggesting ischemia or infarction. A left ventricular aneurysm was found in 14 of the 25 patients, segmental akinesia or hypokinesia in 5, and diffuse hypokinesia in 3. Programmed ventricular stimulation performed in 13 patients induced sustained ventricular tachycardia in 9 and nonsustained ventricular tachycardia in 2. Actuarial survival (mean +/- SE) after four years for the entire group was 56 +/- 12 percent; it was 32 +/- 16 percent among those with global left ventricular dysfunction, and 78 +/- 14 percent among those without such dysfunction (P = 0.03). Only patients with left ventricular dysfunction or an aneurysm died (four-year survival, 45 +/- 14 percent, as compared with 100 percent for the remaining patients; P = 0.0002). Heart failure and left ventricular aneurysm or dysfunction were the only independent predictors of death. Nine patients required permanent pacemakers. CONCLUSIONS. In the United States, Chagas' heart disease commonly mimics coronary artery disease or idiopathic dilated cardiomyopathy. The prognosis is poor for patients with heart failure or left ventricular aneurysm or dysfunction. The disease may be underdiagnosed in the United States.     

Characterization of an Immunodominant Antigenic Epitope from Trypanosoma cruzi as a Biomarker of Chronic Chagas' Disease Pathology

Nowadays, the techniques available for chronic Chagas'' disease diagnosis are very sensitive; however, they do not allow discrimination of the patient''s clinical stages of the disease. The present paper describes that three out of the five different repeats contained in the Trypanosoma cruzi TcCA-2 membrane protein (3972-FGQAAAGDKPPP, 6303-FGQAAAGDKPAP, and 3973-FGQAAAGDKPSL) are recognized with high sensitivity (>90%) by sera from chronic Chagas'' disease patients and that they are not recognized by sera from patients in the acute phase of the disease. A total of 133 serum samples from chagasic patients and 50 serum samples from healthy donors were tested. In addition, sera from 15 patients with different autoimmune diseases, 43 serum samples from patients suffering an infectious disease other than Chagas'' disease, and 38 serum samples from patients with nonchagasic cardiac disorders were also included in this study. The residue 3973 peptide shows a specificity of >98%, as it is not recognized by individuals with autoimmune and inflammatory processes or by patients with a nonchagasic cardiomyopathy. Remarkably, the levels of antibody against the 3973 epitope detected by the sera from Chagas'' disease patients in the symptomatic chronic phase, involving cardiac or digestive alterations, are higher than those detected by the sera from Chagas'' disease patients in the indeterminate phase of the disease. It is suggested that the diagnostic technique described could also be used to indicate the degree of pathology. The amino acids F, Q, and DKP located in the peptide at positions 1, 3, and 8 to 10, respectively, are essential to conform to the immunodominant antigenic epitope.     

Deep sequencing reveals multiclonality and new discrete typing units of Trypanosoma cruzi in rodents from the southern United States

Background/purposeThe parasitic protozoa Trypanosoma cruzi, is widely distributed throughout the Americas. We explored the nature of T. cruzi infection in small rodents from New Orleans (LA, USA), an enzootic region of the parasite in North America.MethodsWe characterized the full complement of discrete typing units (DTUs) in rodent hosts through next-generation metabarcoding, as conventional PCR and Sanger sequencing approaches only detect the dominant genotype in biological samples. We assayed DTU diversity in tissue samples from 6 T. cruzi PCR positive rodents. The intergenic region of the mini-exon gene was amplified and sequenced on a MiSeq platform. A total of 141 sequences were aligned using Muscle, and TCS networks were constructed to identify DTUs in the samples.ResultsWe detected distinct and varying assemblages of DTUs in the rodent hosts. Highly diverse DTU assemblages were detected, with 6–32 haplotypes recovered per individual, spanning multiple DTUs (TcI,TcII, TcIV, TcV and TcVI). Haplotypes varied in frequencies from 82% to less than 0.1%. DTU composition varied according to the tissue analyzed. Rural and urban rodents carried similarly diverse DTU assemblages, though urban rodent species tended to harbor more haplotypes than their sylvatic counterparts.ConclusionOur results affirm that mammalian hosts can concurrently harbor a diverse complement of parasites, and indicate that there is greater diversity of T. cruzi DTUs present in North America than previously thought. Further investigation is warranted to understand the role of commensal rodents as a reservoir for T. cruzi in sylvatic and peridomestic environments.     

Evaluation of a recombinant Trypanosoma cruzi mucin-like antigen for serodiagnosis of Chagas' disease

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and is one of the most important endemic problems in Latin America. Lately, it has also become a health concern in the United States and Europe. Currently, a diagnosis of Chagas' disease and the screening of blood supplies for antiparasite antibodies are achieved by conventional serological tests that show substantial variation in the reproducibility and reliability of their results. In addition, the specificity of these assays is curtailed by antigenic cross-reactivity with sera from patients affected by other endemic diseases, such as leishmaniasis. Here we used a highly sensitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) to evaluate a recombinant protein core of a mucin-like molecule (termed trypomastigote small surface antigen [TSSA]) for the detection of specific serum antibodies in a broad panel of human sera. The same samples were evaluated by CL-ELISA using as the antigen either a mixture of native T. cruzi trypomastigote mucins or an epimastigote extract and, for further comparison, by conventional serologic tests, such as an indirect hemagglutination assay and indirect immunofluorescence assay. TSSA showed ～87% sensitivity among the seropositive Chagasic panel, a value which was increased up to >98% when only parasitologically positive samples were considered. More importantly, TSSA showed a significant increase in specificity (97.4%) compared to those of currently used assays, which averaged 80 to 90%. Overall, our data demonstrate that recombinant TSSA may be a useful antigen for the immunodiagnosis of Chagas' disease.     

Cell-mediated reactivity against human and Trypanosoma cruzi antigens according to clinical status in Chagas' disease patients

The presence of cellular reactivity against homologous tissues and subcellular fractions of Trypanosoma cruzi was investigated in Chagas' disease patients (CDP). CDP were grouped in asymptomatic (CDP-1) and with probable (CDP-2) and overt (CDP-3) cardiomyopathy. Healthy and non-Chagasic cardiomyopathic subjects were studied as controls. Lymphoproliferative reactions against heart tissue extracts were detected in 42% of 72 CDP studied, with similar prevalence of positive reactions in all groups, and correlated with reactivity to both liver and kidney homologous tissues (P less than 0.001). These results confirm the existence of cellular immune reactivity against tissues in CDP, and indicate the lack of organ specificity of this reaction as well as the absence of relation with the clinical state of patients. Cellular reactivity to subcellular fractions of T. cruzi showed a definite pattern according to the clinical status of CDP. Although prevalence of T. cruzi stimulation appeared similar in all groups (70% in CDP-1, 82% in CDP-2 and 75% in CDP-3), CDP-3 showed a significantly higher reactivity to flagellar (69%) and cytosol (63%) fractions than CDP-1 (38 and 27%, respectively). These findings suggest a variable modulation of immune response according to the clinical state of T. cruzi infected subjects.     

Trypanosoma cruzi infection enhances polyreactive antibody response in an acute case of human Chagas' disease

The kinetics of antibody response in an acute case of human Chagas' disease was investigated. Hypergammaglubulinaemia appeared at day 17 of infection, and persisted after 66 days of infection, at which time parasitaemia became undetectable. Titration of immunoglobulins showed that the three principal isotypes were involved in the response, emphasizing polyclonal B cell activation. Total IgA was detected before total IgM, and the killer before total IgG. High titres of autoantibodies were found among IgM and IgG subclasses. IgA was also the first isotype to be detected among specific anti-Trypanosoma cruzi antibodies. However, the maximal parasite antibody response was attained after 30 days of infection for all isotypes. With regard to possible cross-reactivity between molecules of host and parasite, adsorption experiments on T. cruzi-specific immunosorbent were designed. Specific antibodies, present in the eluates, also recognized natural antigens, especially laminin. In order to characterize the α-galactose epitope of laminin, adsorption experiments on sheep erythrocytes were performed, and revealed the possible presence of another epitope on the glycoprotein. Our results indicate that in the case of Chagas' disease investigated here, polyclonal activation occurred; moreover, they suggest that molecular mimicry may play a role by increasing autoantibodies, probably via a parasite-driven mechanism.     

Chagas' disease. Immunotoxin inhibition of Trypanosoma cruzi release from infected host cells in vitro

Virulent tissue culture-derived Trypanosoma cruzi trypomastigotes were readily killed by immune IgG-ricin A chain conjugates (ITR) in vitro. Forty micrograms of ITR immobilized 10(6) trypomastigotes after 48 hours of incubation at 37 degrees C. ITR showed antibody specificity and 125I-labeled anti-T. cruzi IgG bound to parasitized host cells 9-fold more than to nonparasitized host cells. The degree of specificity was evaluated further in experiments in which 10 micrograms of ITR showed 78% inhibition of [3H]thymidine incorporation by T. cruzi. In contrast, nonimmune IgG-ricin A chain conjugate neither immobilized nor inhibited [3H]thymidine incorporation by the parasite. Furthermore, 20 micrograms of ITR significantly inhibited T. cruzi trypomastigote release from infected host cells and thus prevented reinfection of other cells in vitro.     

Chronic Chagas' disease cardiomyopathy patients display an increased IFN-gamma response to Trypanosoma cruzi infection

One-third of all Trypanosoma cruzi -infected patients eventually develop chronic Chagas' disease cardiomyopathy (CCC), a particularly lethal inflammatory dilated cardiomyopathy, where parasites are scarce and heart-infiltrating mononuclear cells seem to be the effectors of tissue damage. Since T. cruzi is a major inducer of interleukin-12 production, the role of inflammatory cytokines in the pathogenesis of CCC was investigated. We assayed cytokine production by peripheral blood mononuclear cells (PBMC) from CCC and asymptomatic T. cruzi -infected (ASY) individuals, as well as by T cell lines from endomyocardial biopsies from CCC patients. PBMC from CCC and ASY patients produced higher IFN-gamma levels than normal (N) individuals in response to B13 protein and phytohaemagglutinin PHA; IFN-gamma high responders (> or =1 ng/ml) were 2-3 fold more frequent among CCC patients than ASY individuals. Conversely, IL-4 production in response to the same stimuli was suppressed among T. cruzi -infected patients. The frequency of PHA-induced IFN gammaproducing cells on PBMC was significantly higher among CCC than ASY and N individuals. IFN-gamma and TNF-alpha were produced by ten out of ten PHAstimulated T cell lines from CCC patients; IL-2 and IL-10 were produced by four out of ten and one out of ten lines, respectively; IL-4, IL-1alpha, IL-1beta, IL-6 and IL-12 were undetectable. Our results suggest that CCC and ASY patients may respond differentially to the IFN-gamma-inducing stimulus provided by T. cruzi infection. Given the T(1)-type cytokine profile of heart-infiltrating T cell lines from CCC patients, the ability to mount a vigorous IFN-gamma response may play a role on the differential susceptibility to CCC development.     

Excretory-secretory antigens of Trypanosoma cruzi are potentially useful for serodiagnosis of chronic Chagas' disease

The reactivities of sera from chronic chagasic patients against the trypomastigote excreted-secreted antigens (TESA) of Trypanosoma cruzi strains with different biodemes were analyzed by TESA-blot and TESA-enzyme-linked immunosorbent assay (ELISA). Although both tests presented high sensitivity and specificity, TESA-ELISA is more appropriate for screening a larger number of samples.     

Purified excreted-secreted antigens from Trypanosoma cruzi trypomastigotes as tools for diagnosis of Chagas' disease

There is currently no "gold standard" test for the diagnosis of late-stage Chagas' disease. As a result, protection of the blood supply in areas where Chagas' disease is endemic remains problematic. A panel of 709 serum samples from subjects with confirmed Chagas' disease (n = 195), healthy controls (n = 400), and patients with other parasitic diseases (n = 114) was used to assess enzyme-linked immunosorbent assays (ELISAs) based on a concentrated extract of excretory-secretory antigens from either Brazil or Tulahuen strain Trypanosoma cruzi trypomastigotes (total trypomastigote excretory-secretory antigens [TESAs]). The total TESA-based assays had excellent overall sensitivity (100%) and specificity (>94%), except for cross-reactivity with Leishmania-infected sera. In an attempt to increase the specificity of the assay, immunoaffinity chromatography was used to purify the TESA proteins (TESA(IA) proteins). By Western blotting, a series of polypeptide bands with molecular masses ranging from 60 to 220 kDa were recognized by pooled sera positive for Chagas' disease. An ELISA based on TESA(IA) proteins had a slightly lower sensitivity (98.6%) but an improved specificity (100%) compared to the sensitivity and specificity of the total TESA protein-based ELISAs. A 60-kDa polypeptide was identified as a major contributor to the cross-reactivity with Leishmania. These data suggest the need for field validation studies of TESA- and TESA(IA)-based assays in regions where Chagas' disease is endemic.     

Enzyme-Linked Immunosorbent Assay for Serological Diagnosis of Chagas' Disease Employing a Trypanosoma cruzi Recombinant Antigen That Consists of Four Different Peptides

Serological tests to detect Trypanosoma cruzi antibodies have been used for screening blood donors, for epidemic studies, and for diagnosis of probably infected persons. Among different tests, the enzyme-linked immunosorbent assay (ELISA) with total, semipurified, or synthetic antigens has been widely used, mainly due to its easy automation. Aiming to improve serological studies concerning Chagas' disease, we have developed and evaluated a new test, the TcF-ELISA, using an artificially engineered recombinant antigen, which contains tandem sequences of different T. cruzi-specific peptides. The sensibility of the TcF-ELISA was determined with 101 serum samples from chagasic patients well-defined by clinical and epidemiological criteria. The specificity was determined with 39 serum samples from leishmaniasis or kala-azar patients and 150 serum samples from nonchagasic blood donors from Sao Paulo, Brazil. The TcF-ELISA showed 100% sensitivity and 98.94% of specificity. Compared with conventional ELISA (with semipurified T. cruzi epimastigote antigens), the TcF-ELISA showed advantages; for example, it distinguishes better between reagent and nonreagent serum and provides better precision and a lower occurrence of leishmaniasis cross-reactions. Our studies demonstrate high reproducibility between two different lots of the TcF ELISA and its applicability for the serological diagnosis of Chagas' disease.     

Enzyme-Linked Immunoassay Using Recombinant trans-Sialidase of Trypanosoma cruzi Can Be Employed for Monitoring of Patients with Chagas' Disease after Drug Treatment

trans-Sialidase is an enzyme present on the surface of Trypanosoma cruzi and is an important antigen recognized by sera from patients with Chagas' disease. In the present study we investigated whether the benznidazole treatment of patients with Chagas' disease induced changes in the reactivity of serum toward a recombinant form of trans-sialidase in order to develop an assay for monitoring of patients after treatment for Chagas' disease, which is needed at Chagas' disease control centers. By using an enzyme-linked immunosorbent assay containing a recombinant protein corresponding to the catalytic domain of trans-sialidase, we found that the antigen had a high specificity for sera from untreated patients with Chagas' disease. Sera from healthy individuals or patients with active visceral leishmaniasis minimally cross-reacted with the antigen. Anti-trans-sialidase immunoglobulin was detected in 98% of 151 untreated patients with Chagas' disease. Of these, 124 patients were treated for 60 days with benznidazole (5 mg/kg of body weight/day), and their sera were assayed for reactivity with the recombinant trans-sialidase. By using this methodology, three groups of patients could be established. The first group (60 patients), which was considered to have been successfully treated, showed no reactivity after treatment. The second group (46 patients) still showed signs of infection, and after treatment their sera recognized trans-sialidase, but with reduced titers. The third group (18 patients) was considered to be resistant to drug treatment, and their sera presented identical reactivities before and after treatment. These results suggest that determination of the absence of antibodies to recombinant trans-sialidase in treated patients by the present assay is indicative of treatment success, while the presence of antibodies may indicate the persistence of infection. Therefore, this method may be useful for the diagnosis and monitoring of patients undergoing benznidazole treatment.     

Trypanosoma cruzi modulates the profile of memory CD8+ T cells in chronic Chagas' disease patients

We present a cross-sectional analysis of the maturation and migratory properties of the memory CD8(+) T cell compartment, in relation to the severity of heart disease in individuals with chronic Trypanosoma cruzi infection removed from endemic areas for longer than 20 years. Subjects with none or mild heart involvement were more likely to mount T. cruzi-specific memory IFN-gamma responses than subjects with more advanced cardiac disease, and the T. cruzi-specific CD8(+) T cell population was enriched in early-differentiated (CD27(+)CD28(+)) cells in responding individuals. In contrast, the frequency of CD27(+)CD28(+)CD8(+) T cells in the total memory CD8(+) T cell population decreases, as disease becomes more severe, while the proportion of fully differentiated memory (CD27(-)CD28(-)) CD8(+) T cells increases. The analysis of CCR7 expression revealed a significant increase in total effector/memory CD8(+) T cells (CD45RA(-)CCR7(-)) in subjects with mild heart disease as compared with uninfected controls. Altogether, these results are consistent with the hypothesis of a gradual clonal exhaustion in the CD8(+) T cell population, perhaps as a result of continuous antigenic stimulation by persistent parasites.     

Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas' disease

A Trypanosoma cruzi antigen which is shed into the culture medium by the trypomastigote stage of the parasite and detected in blood of acutely infected mice was cloned and characterized. We designate this antigen shed acute phase antigen (SAPA). Five protein bands with apparent molecular masses ranging from 160 to 200 kDa were detected by immunoblotting of plasma from infected mice and in supernatants of cultured trypomastigotes upon reaction with antibodies against SAPA. A serum obtained from a patient acutely infected with Chagas' disease revealed a similar set of polypeptides in supernatants of cultured trypomastigotes when tested by immunoblotting. SAPA seems thus to be a major shed protein during the acute period of the disease. Twenty-six of 28 sera from human acute cases of Chagas' disease tested reacted with SAPA. Conversely, only 8-10% of sera from chronic cases of the disease contained detectable levels of antibody against SAPA. Sera from rabbits infected with six different parasite strains all contained antibodies against SAPA. Antibodies against SAPA are detectable 15 days after the manifestation of acute Chagas' disease symptoms in humans and 15 days post-infection in sera from mice and rabbits. The nucleotide sequence of a genomic clone encoding the 3' end of the SAPA gene revealed the presence of 14 tandemly arranged 12-amino acid-long repeats. A 39-amino acid-long region that is very hydrophobic precedes the stop codon. Due to its early appearance it might be possible to design diagnostic assays which are based on SAPA for identification of recently infected cases of Chagas' disease.     

Protective immunity induced by a Trypanosoma cruzi soluble extract antigen in Experimental Chagas' Disease. Role of Interferon γ

CBA/J mice can be protected against lethal infection with Trypanosoma cruzi by treatment using T. cruzi soluble extract antigen (TCSE). In vivo administration of TCSE (400 μg/mouse) into naive mice increased the cellular proliferative response to Con A and elevated the levels of IFN-γ. The production of IFN-γ was extremely important in controlling the replication of the parasite since the protective activity of TCSE was completely abrogated by in vivo treatment with an anti IFN-γ neutralizing antibody. These results suggest that depending on the level, cytokine production results in the control of replication of the parasite in experimental Chagas'disease.